Since 1989, cloning of vascular or platelet adhesive proteins, now termed "selectins," has led to focused attempts to identify carbohydrate epitopes which are expressed on leukocytes (Varki, A., 1994, Proc. Natl. Acad. Sci. U.S.A. 91:7390-7397; Lasky, L. A., 1995, Ann. Rev. Biochem. 64:113-139) and function as targets of selectin-dependent "rolling" and adhesion of leukocytes on activated endothelial cells, followed by transendothelial migration. This mechanism plays a central role in inflammatory responses (Lasky, L. A., 1995, Ann. Rev. Biochem. 64:113-139). Such epitopes are involved in recruitment of the cells to inflammatory sites following infection or wounding. Currently, sialosyl-Le.sup.x (SLe.sup.x) is generally believed to be the target epitope of E-selectin binding, based on the following claims: (i) Human leukocytes, leukemic leukocytes, and leukemic cell lines (e.g., HL60 and U937 cells), but not non-human leukocytes, express SLe.sup.x. This claim was based on strong reactivities of these types of cells with mAbs believed to be directed to SLe.sup.x (Ito et al., 1994, Glycoconj. J. 11:232-237). These SLe.sup.x -expressing cells adhere to activated endothelial cells or platelets which express E- or P-selectin (Phillips et al., 1990, Science 250:1130-1132; Polley et al., 1991, Proc. Natl. Acad. Sci. U.S.A. 88:6224-6228). (ii) Chinese Hamster Ovary (CHO) cells expressing sialosyl type 2 chain do not adhere to E-selectin, whereas transfectants of these cells with fucosyltransferase III cDNA do adhere to E-selectin (Lowe et al., 1991, J. Biol. Chem. 266:17467-17477). (iii) E-selectin-dependent adhesion of SLe.sup.x -expressing cells to activated ECs is inhibited by liposomes containing SLe.sup.x GSLs, or by oligosaccharides with terminal SLe.sup.x structure (Phillips et al., 1990, Science 250:1130-1132; Polley et al., 1991, Proc. Natl. Acad. Sci. U.S.A. 88:6224-6228; Handa et al., 1991, Biochem. Biophys. Res. Commun. 181:1223-1230).
These observations have encouraged acceptance of the idea that SLe.sup.x is the epitope to which E-selectin binds. E- and P-selectin also bind to SLe.sup.a, the positional isomer of SLe.sup.x (Handa et al., 1991, Biochem. Biophys. Res. Commun. 181:1223-1230; Berg et al., 1991, J. Biol. Chem. 266:14869-14872; Takada et al., 1991, Biochem. Biophys. Res. Commun. 179:713-719); however, SLe.sup.a is absent in leukocytes and is not considered to be a physiologic epitope of selectins for hematopoietic cells. There has been no systematic characterization of SLe.sup.x -containing gangliosides present in neutrophils and HL60 cells, nor any unambiguous demonstration that SLe.sup.x is the major epitope present in N-linked or O-linked glycoprotein side chains in normal or leukemic leukocytes or cell lines derived therefrom.
It is reported that, in an IgG immune complex model of rat with neutrophil-mediated and E-selectin-dependent lung injury, SLe.sup.x provides protective effects against inflammatory vascular injury (Mulligan et al., 1993, J. Exp. Med. 178:623-631).
However, it is also reported that, from the results of immunostaining by antibodies and of indirect binding assay to E- or P-selectin affixed on plate, of human neutrophil (polymorphonuclear leukocytes; PMN), only human PMN and promyelogenous leukemia HL60 cell expressed SLe.sup.x and other lacto-series epitopes, such as Le.sup.x or Le.sup.y, but no other mammalian PMN, such as PMN of baboon, macaque, pig, rabbit, rat, guinea pig and hamster (Ito et al., 1994, Glycoconj. J. 11:232-237). And that the E-selectin ligand saccharide sequences obtained from mouse kidney and murine leukocyte are identified as,
Gal.beta.4GlcNAc.beta.6GalNAc.beta.3Gal.alpha.4Gal.beta.4Glc.beta.1Cer 3 3 Fuc.alpha.1 Gal.beta.1 - and - Gal.beta.3GlcNAc.beta.3Gal 3 4 NeuAc.alpha.2 Fuc.alpha.1
(Osanai et al., 1996, Biochem. Biophys. Res. Commun. 218:610-615).
These reports showed that ligands for selectin of mammals other than human beings are not SLe.sup.x. And hitherto certified results on anti-inflammatory effects obtained by using animal models have become questionable.
Further, it is disclosed that, when in vitro, liposomes containing the glycolipid;
Gal.beta.4GlcNAc.beta.3Gal.beta.4GlcNAc.beta.3Gal.beta.4Glc.beta.1Cer 3 3 3 NeuAc.alpha.2 Fuc.alpha.1 Fuc.alpha.1
are added to activated endothelial cells and thereto HL60 cells are added, binding of HL60 cells to activated endothelial cells are selectively blocked (WO91/19501 and WO91/19502).
Further, it is reported that glycolipids extracted from leukocytes of patients with chronic myelogenous leukemia was either absorbed to polyvinyl chloride microtiter wells or resolved on TLC plates, screened by binding to COS cells expressing endothelial leukocyte adhesion molecule-1 (ELAM-1) and analyzed structurally, so that detected was the glycolipid below:
Gal.beta.4GlcNAc.beta.3Gal.beta.4GlcNAc.beta.3Gal.beta.4GlcNAc.beta.3G al.beta.4Glc.beta.1Cer 3 3 NeuAc.alpha.2 Fuc.alpha.1
(Proc. Natl. Acad. Sci. U.S.A. 88:1138-1142 [1991]).
Further, Stroud et al. (Biochem. Biophys. Res. Commun. 209: 777-787 [1995]) reported that the following glycolipids,
Gal.beta.4GlcNAc.beta.3Gal.beta.4GlcCer, 3 3 NeuAc.alpha.2 Fuc.alpha.1 - Gal.beta.4GlcNAc.beta.3Gal.beta.4GlcNAc.beta.3Gal.beta.4GlcCer 3 3 NeuAc.alpha.2 Fuc.alpha.1 - and - Gal.beta.4GlcNAc.beta.3Gal.beta.4GlcNAc.beta.3Gal.beta.4GlcC er 3 3 3 NeuAc.alpha.2 Fuc.alpha.1 Fuc.alpha.1
commonly found in solid tumor cells and tissues does not exist in human neutrophils and HL60 cells and that the following glycolipids,
#1 Gal.beta.4GlcNAc.beta.3Gal.beta.4GlcNAc.beta.3Gal.beta.4GlcNAc.beta.3 Gal.beta.4GlcNAc.beta.3Gal.beta.4GlcCer, 3 3 NeuAc.alpha.2 Fuc.alpha.1 - #2 Gal.beta.4GlcNAc.beta.3Gal.beta.4GlcNAc.beta.3Gal.beta.4GlcNAc.b eta.3Gal.beta.4GlcNac.be ta.3Gal.beta. 4GlcCer, 3 3 NeuAc.alpha.2 Fuc.alpha.1 - #3 Gal.beta.4GlcNAc.beta.3Gal.beta.4GlcNAc.beta.3Gal.beta.4GlcNAc.b eta.3Gal.beta.4GlcNAc.be ta.3Gal.beta.4GlcNac.bet a.3Gal.beta.4GlcCer, 3 3 NeuAc.alpha.2 Fuc.alpha.1 - #4 Gal.beta.4GlcNAc.beta.3Gal.beta.4GlcNAc.beta.3Gal.beta.4GlcNAc.b eta.3Gal.beta.4GlcNAc.be ta.3Gal.beta.4GlcCer 3 3 3 NeuAc.alpha.2 Fuc.alpha.1 Fuc.alpha.1 - and - #5 Gal.beta.4GlcNAc.beta.3Gal.beta.4GlcNAc.beta.3Gal.beta.4GlcNAc. beta.3Gal.beta.4GlcNAc.b eta.3Gal.beta.4GlcCer 3 3 3 3 NeuAc.alpha.2 Fuc.alpha.1 Fuc.alpha.1 .+-.Fuc.alpha.1
were extracted from human neutrophils and HL60 cells, developed on TLC and placed into contact with E-selectin expressing CHO cells to detect adhesion, which proved that these cells adhered to the glycolipid #4 including very little amount of #5.
On the other hand, since the rolling-type adhesion between the selectins on vascular endothelium and the oligosaccharide ligands of leukocytes participates in the initiation of the inflammatory response, it is expected to protect from influx of leukocytes into the tissue sites of inflammation and localized damage to endothelium by activated neutrophils via an inhibition of leukocyte rolling along endothelium (Lasky, 1995, Ann. Rev. Biochem. 64:113-139).
The currently known E-selectin ligand compounds were selected and proved to be effective under conditions without any shear stress, not taking into consideration the abovementioned rolling phenomena really occurring in human body. Therefore, these compounds should not be a real E-selectin ligand material. They could control neither E-selectin dependent rolling and adhesion of leukocytes along E-selectin expressing cells, such as endothelium, which is activated in living body nor human inflammation specifically.
It is reported that E-selectin expressing CHO cells tethered under a shear stress of 0.73 dyne/cm.sup.2 along the solid phase affixed with SLe.sup.x via egg lecithin phosphatidylcholine (abbreviated as PC). The solid phase used for this experiment was prepared by adding 3 .mu.l of SLe.sup.x (dissolved at 1 .mu.g/ml in 20:1 methanol:butanol solution containing 4 .mu.g/ml PC) to the area having a diameter of 4 mm and drying, whereby, based on the amount added to said solid phase, 15% of SLe.sup.x was affixed via PC to the solid phase (J. Immunol. 154:5356-5366 (1995)). However, as mentioned before, not existing in human neutrophil, SLe.sup.x could not control human inflammation safely and specifically.
A recent report characterized monosialogangliosides of HL60 cells and human neutrophils that bind (or do not bind) to E-selectin under static conditions (Stroud et al., 1995, Biochem. Biophys. Res. Comm. 209:777-787; Stroud et al., 1996, Biochemistry 35:758-769). There was no SLe.sup.x structure, with or without internal fucosylation, having &lt;10-sugar monosaccharide units as poly-LacNAc core structure (Stroud et al., 1996, Biochemistry 35:758-769). All the E-selectin binding fractions had .alpha.2.fwdarw.3 sialosylation at the terminal Gal and two or more .alpha.1 .fwdarw.3 fucosylations at internal GlcNAc other than the penultimate (Stroud et al., 1995, Biochem. Biophys. Res. Comm. 209:777-787; Stroud et al., 1996, Biochemistry 35:770-778). These binding fractions were collectively termed "myeloglycan." There was an extremely minor component of poly-LacNAc having SLe.sup.x terminus with .alpha.1 .fwdarw.3 fucosylation at internal GlcNAc. It was concluded that the major E-selectin binding site in human neutrophils and HL60 cells is myeloglycan type rather than SLe.sup.x -containing glycan. None of the myeloglycan or poly-LacNAc SLe.sup.x structures examined showed P-selectin binding (Stroud et al., 1996, Biochemistry 35:758-769; Stroud et al., 1996, Biochemistry 35:770-778).